Method for chromosomal rearrangement by consecutive gene targeting of two recombination substrates to the deletion endpoints

ABSTRACT

The present invention involves the creation of defined chromosomal deficiencies, inversions and duplications using Cre recombinase in ES cells transmitted into the mouse germ line. These chromosomal reconstructions can extend up to 3-4 cM. Chromosomal rearrangements are the major cause of inherited human disease and fetal loss. Additionally, translocations and deletions are recognized as major genetic changes that are causally involved in neoplasia. Chromosomal variants such as deletions and inversions are exploited commonly as genetic tools in organisms such as Drosophila. Mice with defined regions of segmental haploidy are useful for genetic screening and allow accurate models of human chromosomal diseases to be generated.

The present invention was made utilizing funds of the United StatesGovernment. The U.S. Government is entitled to certain rights under thisinvention.

This application claims the benefit of U.S. Provisional Application Ser.No. 60/020,620, filed Jun. 26, 1996.

BACKGROUND OF THE INVENTION

1. Technical Field of the Invention

The present invention involves the creation of defined chromosomaldeficiencies, inversions and duplications using Cre recombinase inembryonic stem cells and transmitted into the mouse germ line. In thepresent invention, these chromosomal reconstructions can extend up to3-4 cM. Chromosomal rearrangements are the major cause of inheritedhuman disease and fetal loss. Further, chromosomal translocations anddeletions are recognized as major genetic changes that are causallyinvolved in neoplasia. Chromosomal variants such as deletions andinversions are exploited commonly as genetic tools in diploid organismssuch as Drosophila. In diploid organisms, such deficiencies areexploited in genetic screens because a small portion of the genome isfunctionally hemizygous. Thus, a mutation which would normally berecessive and masked by the wildtype allele in a diploid context will bedominant and detectable in the haploid state. In the mouse, deficiencieshave not, up to now, been available generally; thus, screens forrecessive mutations are nonexistent or particularly cumbersome. However,the present invention provides methods to engineer mice and cell lineswith defined regions of segmental haploidy. Such mice are useful forgenetic screening and provide accurate models of human chromosomaldiseases.

2. The Prior Art

Inherited chromosomal rearrangements such as inversions, duplicationsand deficiencies are responsible for a significant fraction of humancongenital disease. Chromosomal changes also occur somatically and areassociated with neoplastic disease. Defining the causal geneticalteration in a region of the genome associated with chromosomalrearrangements can be relatively straightforward if the affected genelies in the breakpoint of an inversion or translocation. However, incases of duplications and deficiencies, the specific genetic lesion(s)associated with pathological chromosomal changes are much harder toidentify. Still, the generation of animal models that accuratelyrecapitulate the genetic lesion would facilitate the study of diseaseand could be very helpful in the efforts to dissect specificgene-function relationships in multigene syndromes.

In diploid organisms such as Drosophila, chromosomal deficiencies arecommonly exploited in genetic screens because a small portion of thegenome is functionally hemizygous. Thus, a mutation which would berecessive and masked by the wildtype allele in the diploid context willbe dominant and therefore readily detectable in the haploid state. Inthe mouse, deficiencies are not available generally. Despite the limitednumber of deficiencies available in the mouse, the potential for thedetailed analysis of a genetic interval using these deficiencies hasbeen demonstrated clearly. See Holdener-Kenny, et al., BioEssays,14:831-39 (1992), which is hereby incorporated by reference.

Deficiencies that are available currently in the mouse genome weregenerated at random using ionizing irradiation. Although conventionalgene targeting technology in embryonic stem (ES) cells can generatevirtually any type of mutation, including deletions of up to 20 kb, ithas not been possible to delete substantially larger fragments by usingstandard methodology. Likewise, the technology required to constructlarge inversions and duplications has not been established.

One mechanism by which chromosomes may be engineered is by the use ofCre recombinase. Cre recombinase has been used in mammalian cell linesand in vivo to delete or invert sequences between the 34 base pairrecognition sequences, loxP sites, placed a few kb apart on the samechromosome. The recombination is initiated by Cre proteins which bind to13-bp inverted regions in the loxP sites and promote synapses or joiningof a pair of sites. Next, the Cre proteins catalyze strand exchangebetween the pair of sites within an asymmetric 8-bp central spacersequence by concerted cleavage and rejoining reactions, involving atransient DNA-protein covalent linkage. Smith, et al., Nature Genetics,9:376-385 (1995); Gu, et al., Science, 265:103-06 (1994) and Sauer,Nucl. Acids Res., 17:147-61 (1989) (both of these references are herebyincorporated by reference). Additionally, recombinases have been used toinduce mitotic recombination between homologous and non-homologouschromosomes in Drosophila, plants and mammalian cells. Embryonic stemcell technology has become a powerful tool for defining the function ofmammalian genes, but mainly has been restricted to the mutation ofsingle genes. Replacement vectors have been used to construct deletionsof up to 19 kb; however, utilizing the same strategy to construct largerdeletions (>60 kb) has not been successful. In the present invention,the generation and direct selection of deletions, duplications andinversions, ranging from 90 kb to 3-4 cM, in ES cells is demonstrated.

SUMMARY OF THE INVENTION

The method of the present invention is based on consecutive genetargeting of two recombination substrates to the deletion endpoints andthe subsequent induction of recombination mediated by the Crerecombinase. This method generates a positive selectable marker allowingfor the direct selection of clones with the desired chromosomestructures. Despite the multitude of steps involved in generating theserearrangements in ES cells, deletion and duplication alleles have beentransmitted into the mouse genome.

One object of the present invention is a method for causing alarge-scale chromosomal rearrangement by first deleting a portion ofgenetic material.

An additional object of the present invention is a targeting vectorsystem capable of inserting into two endpoint regions constraining adesired chromosomal deletion.

Thus in accomplishing the foregoing objects, there is provided inaccordance with one aspect of the present invention a method fordeleting a selected region of genetic material in cells comprising thesteps of: inserting a first selection cassette at a 5' end of saidselected region using conventional gene targeting methods, said firstselection cassette comprising a first selectable marker, a first loxPrecombination site, and a first portion of a second selectable marker;selecting cells expressing said first selectable marker; inserting asecond selection cassette at a 3' end of said selected region usingconventional gene targeting methods, said second selection cassettecomprising a third selectable marker, a second loxP recombination site,and a remaining portion of said second selectable marker; selectingcells expressing said third selectable marker; expressing Crerecombinase to produce recombination between said first and second loxPsites; and selecting cells expressing said second selectable marker.

Specific embodiments of the above method can include a puromycinresistance gene as the first selectable marker, a functional Hprt geneas the second selectable marker, and a neomycin resistance gene as thethird selectable marker. Numerous other selectable markers will work,their presence in the particular deletion strategy is merely to aid cellselection. In other preferred embodiments, the first selectable markeris a puromycin resistance gene. In still other preferred embodiments,the second selectable marker is a functional Hprt gene. And in stillother preferred embodiments, the third selectable marker is a neomycinresistance gene.

In still other preferred embodiments, the cells referred to above areembryonic stem cells, though significant, they need not be stem cells.In other preferred embodiments, the cells are embryonic stem cells, andsaid cells develop into mice. And in yet other preferred embodiments,the cells are embryonic stem cells, and said cells are maintained ascell lines.

In yet another preferred embodiment, a viral vector is used to replaceeither or both native sequences of DNA. In one embodiment, this virus isa retrovirus. In yet another embodiment, the viral vector referred toabove has a provirus structure comprising a cassette in turn comprising:an hprtΔ5' cassette, a loxP site, and a puromycin resistance gene.

In yet another particularly preferred embodiment, the method fordeleting a portion of chromosomal material in cells wherein thetargeting vectors are a first targeting vector for replacing said firstnative sequence of DNA at said 5' end, comprising: a genomic insertcloned into the vector of about 7.5 kb; a tyrosinase minigene; a Neo^(r)gene; a 5' hprt fragment; and a loxP site embedded into said hprtfragment; and a second targeting vector for replacing said second nativesequence of DNA at said 3' end, comprising: a genomic insert cloned intothe vector of about 8.5 kb; a K14-Agouti gene; a Puro^(r) gene; a 3'hprt fragment; and a loxP site embedded into said hprt fragment.

In one particularly preferred embodiment of the second aspect of thepresent invention, there is provided a replacement vector systemcomprising a first targeting vector for replacing said first nativesequence of DNA at said 5' end, comprising a genomic insert cloned intothe vector of about 7.5 kb; a tyrosinase minigene; a Neo^(r) gene; a 5'hprt fragment; and a loxP site embedded into said hprt fragment; and asecond targeting vector for replacing said second native sequence of DNAat said 3' end, comprising: a genomic insert cloned into the vector ofabout 8.5 kb; a K14-Agouti gene; a Puro^(r) gene; a 3' hprt fragment;and a loxP site embedded into said hprt fragment.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A through 1E: 1A: depicts the Hprt-loxP minigene cassette; 1B:depicts the hprtΔ5' and hprtΔ3' recombination substrates; 1C: outlinesthe general strategy for Cre-induced, targeted genomic rearrangementsillustrated at the HoxB cluster--only the intrachromosomal pathway isshown; 1D: demonstrates alternative orientations (A or B) of therecombination substrates at the E2DH and Gastrin loci; 1E: showschromosomal alterations induced by Cre recombinase for the differentorientations of the minigenes in cis and trans.

FIGS. 2A through 2E: Shows the results of a Southern blot analysis ofthe chromosomal engineering technology used to delete the HoxB cluster.A-C demonstrates the interpretation of, and D-F shows the actualSouthern blot data from wildtype (wt), double targeted (dt) or HATresistant ES cell clones (lanes 1 and 2). M1 and M2 are HindIII-cut andBstEII-cut lambda DNA molecular weight markers, respectively. Hoxb-1 islocated in a 7.2 kb NheI fragment detected with probe a (2A and 2D; thisallele is present in all of the lanes). Targeting of the hprtΔ3'-neocassette to the Hoxb-1 locus generates a novel 10.2 kb NheI restrictionfragment detected with probe a (2B and 2D dt). Hoxb-9 is located on a 16kb NheI restriction fragment detected with probe b (2A and 2E; thisallele is present in all of the lanes). Targeting of the hprtΔ5'-puro tothe Hoxb-9 gene generates a novel 20.4 kb NheI restriction fragmentdetected with probe b (2B and 2E dt). Cre-induced recombination bringstogether the hprtΔ5' and hprtΔ3' and produces a NheI 18.2 kbdeletion-specific junction fragment detected by both probes a and b (2C,2D 1 and 2). Probe c, located in the deletion region, shows a dosagedifference in Panel 2F, 1 and 2, compared to the wt and dt lanes. Probea is a 0.7 kb RsaI fragment located approximately 3 kb downstream ofHoxb-1 exon 2; probe b is a 1 kb RsaI fragment located approximately 5kb upstream of Hoxb-1 exon 1. P and N represent the puromycin andneomycin selection cassettes.

FIGS. 3A through 3E: 3A: depicts mouse chromosome 11, and the loci usedas endpoints for the chromosome engineering are illustrated. 3B: showsNheI restriction sites (N) and fragment lengths around the E2DH andGastrin loci. 3C: shows chromosome 11 with both the E2DH and Gastrinloci targeted with the hprtΔ5' and hprtΔ3' vectors, respectively. Forclarity, only the cis configuration and the A orientation are shown. 3D:shows the structure of the deletion, duplication and inversion alleles.The sizes of the diagnostic restriction fragments and probes used todetect these alleles are indicated. The deletion, duplication andinversion alleles are derived from the double targeted chromosome in theAA, BB and AB configurations, respectively. 3E: shows the results ofSouthern blots which confirm the structure of the recombinantchromosomes. The probes used with each blot (a, b, c or d) are indicatedbeneath each panel and on the diagrams of the various alleles. The lanesare coded as follows: wildtype (wt), double targeted (dt), deletion(del), duplication (dup) and inversion (inv).

FIGS. 4A through 4C: G₂ trans recombination between homologouschromosomes. 4A: depicts individual sister chromatids from chromosomehomologues still joined at the centromere. One chromosome is illustratedwith hprtΔ5', the neomycin resistance gene (N) used for targeting. Theother homologue was targeted with hprtΔ3' cassette linked to thepuromycin resistance gene (P). Cre-induced recombination between loxPsites on sister chromatids from different homologues is illustrated byan X. B: illustrates the recombinant structure of the sister chromatids.The individual chromatids are numbered 1-4. Chromatid 3 carries thereconstructed Hprt minigene and the deletion. 4C: shows the results ofHAT selection for chromatid 3 which will segregate with either chromatid1 or 2 which carries only the neo or both the neo and puro cassettes,respectively. The chromatid 2+3 segregant carries a duplication anddeletion (genetically balanced) and is indistinguishable from the G₁inter-chromosomal product. The chromatid 1+3 product carries thedeletion and the original single targeted chromosome; this can only havearisen via the G₂ pathway.

FIG. 5: Gene dosage analysis and segregation of the deletion andduplication chromosomes through the mouse germ line are demonstrated:Lane 1: wildtype allele (AB2.2 ES cell line); Lane 2: ES cell clone withthe duplication and deletion (genetically balanced); Lanes 3 to 6:transmission and segregation of the duplication and deletion alleles inthe progeny of a chimeric male constructed from the cells shown in lane2. Lanes 3 and 4 show mice with the deletion, lanes 5 and 6 show micewith the duplication; the increase or decrease in intensity of the 9.0kb fragment relative to the 2.0 kb control fragment is consistent withjunction fragment analysis of the inheritance of the duplication ordeletion alleles from these mice (data not shown). Lanes 7 and 8 showmice from heterozygous mating homozygous for the duplication allelewhich is evident from the increased intensity of the 9.0 kb fragment.

FIGS. 6A through 6D: Deletion of two 3-4 cM intervals on mousechromosome 11 is shown. 6A depicts mouse chromosome 11. The shaded barsindicate the intervals which are to be deleted. Hox B, E2DH and Wnt3 arethe loci which serve as the deletion endpoints. SBC (Sporadic BreastCancer) loci are indicated by the black bars. These loci are theputative location of tumor suppressor genes based on the analysis ofloss of heterozygosity in breast cancer. 6B depicts a double-targetedchromosome which has been targeted with the hprtΔ3' cassettes to theHoxb9 and E2DH genes or to the E2DH and Wnt3 genes. The A orientationE2DH-targeted clones were used with the HoxB-E2DH deletion and the Borientation clones were used with the E2DH-Wnt3 deletion. Only theorientations of the hprtΔ5' cassette which give the deletion productsare illustrated. The vertical bars represent NheI sites; the sizes ofthe fragments are indicated and the probes are indicated by shaded boxeslabelled c and d. 6C shows the structure of deletion alleles. DiagnosticNheI fragments for the deletion are indicated. 6D reveals the Southernanalysis that confirms the structure of the alleles in the wildtype(wt), double-targeted (dt) and deletion (dt) clones. Thedeletion-specific junction fragments are indicated by the arrows.

FIG. 7: Schematic representation of a provirus structure, containinghprtΔ5' minigene cassette, a loxP site, and a puromycin resistance gene,for use as a vector in one embodiment of the present invention. Thisparticular vector would insert the loxP site at the 5' end of thechromosome.

FIG. 8: A 3' anchor library that contains the expression cassette 3'hprt, puromycin resistance gene, and k14-agouti gene.

FIG. 9: A 5' anchor library that contains the expression cassettes 5'hprt, neomycin resistance gene, and tyrosinase gene.

FIG. 10: Map of an exemplary 5' endpoint targeting vector automaticallyexcised out of a phage clone isolated from the 5' anchor library.

FIG. 11: Map of an exemplary 3' endpoint targeting vector automaticallyexcised out of a phage clone isolated from the 3' anchor library.

FIG. 12: Map of pG12WT (Wildtype 3' hprt cassette plasmid for makingchromosomal rearrangements). The [SEQ. ID. NO. 2] is identical to pG12[SEQ. ID. NO. 3] except that the mutation in 3' hprt has been fixed.

FIG. 13: Map of a portion of the mouse chromosome 11 showing the generalcomposition of the selection cassettes positioned at the chromosomeendpoints, and the position of the Cre-induced deletion interval, E₂DH-D11Mit199.

FIG. 14: Map of a portion of the mouse chromosome 11 showing the generalcomposition of the selection cassettes positioned at the chromosomeendpoints, and the position of the Cre-induced deletion interval, E₂DH-D11Mit69.

DETAILED DESCRIPTION OF THE INVENTION

It will be apparent readily to one skilled in the art that varioussubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

As used herein, the term "chromosome engineering" means creatingchromosome inversions, duplications or deletions.

As used herein, the term "chromosome deficiency" means a lack of achromosome or portion of a chromosome.

As used herein, the term "chromosome inversion" means reversal of a partof a chromosome so that the genes within that part are in reverse order.

As used herein, the term "chromosome duplication" means an extra,duplicate chromosome or part of a chromosome.

As used herein, the term "ES cell" stands for embryonic stem cells:cells which are derived from early mouse embryos that can be maintainedin an undifferentiated state, but, upon return to the environment of theearly embryo, can contribute to all types of cells in the resultingchimera.

As used herein, the term "Cre-induced recombination" means catalysis ofboth intramolecular and intermolecular recombination by the Cre protein.The Cre protein is a 38 kD protein that recombines DNA between specific,34 bp sequences called loxP sites.

As used herein, the term "interchromosomal recombination" meansrecombination between different chromosomes.

As used herein, the term "intrachromosomal recombination" meansrecombination between regions on the same chromosome.

As used herein, the term "HoxB" refers to a specific gene cluster havingthe same physical distance as a P1 phage and having known structure andorientation.

As used herein, the term "Hoxb-9" refers to a specific gene in the HoxBgene cluster.

As used herein, the term "Hoxb-1" means the 3'-most gene of the HoxBgene cluster.

As used herein, the term "hprt" means hypoxanthinephosphoribosyltransferase.

As used herein, the term "loxP site" means the specific 34 bp sequencerecognized by Cre recombinase.

As used herein, the term "G418 resistance" means having a gene whichconfers resistance to G418.

As used herein, the term "neo resistance" means having a gene whichconfers resistance to G418.

As used herein, the term "puromycin resistance" means having a genewhich confers resistance to puromycin.

As used herein, the term "HAT resistance" means cells resistant to mediacontaining hypoxanthine, aminopterin and thymine. Only cells expressingthe Hypoxanthine phosphoribosyl transferase (Hprt) and thymidine kinasegenes will grow in HAT medium.

As used herein, the term "targeting deletion" means a planned deletioncreated by targeting an area for recombination by insertion of a loxP orother recombination site.

As used herein, the term "germ line transmission" refers to a chimericanimal capable of transmitting a particular trait to its offspring.

As used herein, the term "hemizygous" means genes present only once in agenotype.

As used herein, the term "heterozygous" refers to the state of anorganism having two different alleles at a given locus on homologouschromosomes.

As used herein, the term "homozygous" refers to the state of an organismhaving the same two alleles at a given locus on homologous chromosomes.

As used herein, the term "Gastrin" locus means a gene on mousechromosome 11 which encodes a peptide involved in stimulating acidsecretion in the stomach. See Fuller et al., Molec Endocrinol. 1:306-11(1987).

As used herein, the term "E2DH" locus, also known as 17HSD, is a genewhose product is involved in steroid biosynthesis. This dehydrogenaseconverts estrone to estradiol. See The et al., Molec. Endocrinol.3:1301-06 (1989).

As used herein, the term "Wnt3" locus is a gene which encodes a memberof the wnt family of growth factors. Wnt3 is a target for activation byMMTV which causes mammary tumors. Roelink et al., PNAS 87:4519 (1990).

As used herein, the term "selected region" refers to that particularregion of the chromosome targeted for manipulation (i.e., deletion,inversion, duplication).

In one embodiment of the present invention, a method is disclosed andclaimed for deleting a selected region of genetic material in cellscomprising the steps of: inserting a first selection cassette at a 5'end of said selected region using conventional gene targeting methods,said first selection cassette comprising a first selectable marker, afirst loxP recombination site, and a first portion of a secondselectable marker; selecting cells expressing said first selectablemarker; inserting a second selection cassette at a 3' end of saidselected region using conventional gene targeting methods, said secondselection cassette comprising a third selectable marker, a second loxPrecombination site, and a remaining portion of said second selectablemarker; selecting cells expressing said third selectable marker;expressing Cre recombinase to produce recombination between said firstand second loxP sites; and selecting cells expressing said secondselectable marker.

In particularly preferred embodiments of the present invention, themethod referred to above uses a first selectable marker, a puromycinresistance gene, said second selectable marker is an Hprt gene, and saidthird selectable marker is a neomycin resistance gene.

In another particularly preferred embodiment, the first selectablemarker is a puromycin resistance gene. In yet another particularlypreferred embodiment, the second selectable marker is a functional Hprtgene. In still another preferred embodiment, the third selectable markeris a neomycin resistance gene. In another preferred embodiment, thecells are embryonic stem cells. In still another preferred embodiment,the cells are embryonic stem cells, and said cells develop into mice. Instill another preferred embodiment, the cells are embryonic stem cells,and said cells are maintained as cell lines. In another embodiment ofthe present invention, Cre is transiently expressed Cre. In otherembodiments, it is expressed either inducibly or constitutively.

In a second general embodiment of the present invention, a method isdisclosed and claimed for deleting a selected region of genetic materialin cells comprising the steps of: inserting a first selection cassetteat a 5' end of said selected region using either conventional targetingmethods or a viral vector, said first selection cassette comprising afirst selectable marker, a first loxP recombination site, and a firstportion of a second selectable marker; selecting cells expressing saidfirst selectable marker; inserting a second selection cassette at a 3'end of said selected region using conventional gene targeting methods ora viral vector, said second selection cassette comprising a thirdselectable marker, a second loxP recombination site, and a remainingportion of said second selectable marker; selecting cells expressingsaid third selectable marker; expressing transiently Cre recombinase toproduce recombination between said first and second loxP sites; andselecting cells expressing said second selectable marker.

In one particularly preferred embodiment, the viral vector is aretrovirus. In yet another particularly preferred embodiment, the viralvector has a provirus structure comprising a cassette in turn comprisingan hprtΔ5' cassette, a loxP site, and a puromycin resistance gene. Inyet another particularly preferred embodiment, the viral vector has aprovirus structure comprising a cassette in turn comprising an hprtΔ5'cassette, a loxP site, and a neomycin resistance gene. In still anotherparticularly preferred embodiment, the targeting or viral vectors are afirst vector for inserting said first native sequence of DNA at said 5'end, comprising: a genomic insert cloned into the vector of about 7.5kb; a tyrosinase minigene; a Neo^(r) gene; a 5' hprt fragment; and aloxP site embedded into said hprt fragment; and a second vector forinserting said second native sequence of DNA at said 3' end, comprising:a genomic insert cloned into the vector of about 8.5 kb; a K14-Agoutigene; a Puro^(r) gene; a 3' hprt fragment; and a loxP site embedded intosaid hprt fragment.

In a third general embodiment of the present invention, a replacementvector system, is disclosed and claimed comprising: a first vector forinserting said first native sequence of DNA at said 5' end, comprising:a genomic insert cloned into the vector of about 7.5 kb; a tyrosinaseminigene; a Neo^(r) gene; a 5' hprt fragment; and a loxP site embeddedinto said hprt fragment; and a second vector for inserting said secondnative sequence of DNA at said 3' end, comprising: a genomic insertcloned into the vector of about 8.5 kb; a K14-Agouti gene; a Puro^(r)gene; a 3' hprt fragment; and a loxP site embedded into said hprtfragment.

In a fourth general embodiment of the present invention, there isdisclosed and claimed a method for creating defined chromosomaldeficiencies, deletions, and duplications comprising the steps of:identifying a desired region of a chromosome of interest to be deleted;inserting two native sequences at each endpoint of said region of saidchromosome of interest using a first and a second targeting vector, eachcomprised of one or more selectable markers and a loxP site and an hprtfragment; transiently expressing Cre recombinase to producerecombination between each of two said loxP sites; whereby uponchromosomal rearrangement induced by said Cre recombinase, a functionalHprt expression cassette is reconstructed.

Other and further embodiments, features and advantages will be apparentand the invention more readily understood from a reading of thefollowing Examples and by reference to the accompanying drawings forminga part thereof, wherein the examples of the presently preferredembodiments of the invention are given for the purposes of disclosure.

EXAMPLE A General Strategies

The various chromosomal rearrangements described herein are designedwith strong positive selection for the desired chromosomal change. Verygenerally, this was accomplished by targeting consecutivelycomplementary, overlapping but non-functional hprt-loxP expressioncassettes to the endpoints of a chromosomal interval. Cre expression(either transiently, inducibly, or constitutively) in thesedouble-targeted ES cells induces loxP recombination resulting inchromosomal rearrangements specific to the relative orientation of theloxP sites. Since the loxP sites are imbedded in the hprt minigenefragments, the chromosomal rearrangement will also reconstruct afunctional hprt expression cassette, therefore facilitating directpositive selection for the clones with these alterations.

The use of mouse ES (embryonic stem) cells is preferable, though notrequired, to execute the method the present invention. Use of thesecells would, of course, allow large-scale chromosome manipulation to beintroduced into a germ line, which would in turn facilitate enhancedfunctional study of the mouse genome.

The loxP sites were introduced by conventional gene targeting protocolsor by viral vectors into the endpoints of the region which was to berearranged. Or, one endpoint can be introduced by conventional methods,and the other introduced by a viral vector. To maximize the ability toselect for the rare ES cell clones in which Cre expression hadsuccessfully induced recombination between loxP sites, the individualloxP sites targeted to the endpoints of the chromosomal rearrangementwere imbedded in two complementary but non-functional fragments of anHprt minigene cassette. Recombination between the loxP sites wouldrestore the activity of this cassette, facilitating the direct selectionin HAT media of only those recombinant ES cells with the desiredchromosomal structure (FIGS. 1A and B).

In one particularly preferred embodiment of the present invention, thecomplementary recombination/selection substrates consist of overlapping,but incomplete, pieces of an Hprt minigene with a loxP site in theintron. These minigene fragments are linked to different positiveselection cassettes which are required for selection during genetargeting. The 5' fragment of the loxP-Hprt minigene is linked to aneomycin resistance gene (hprtΔ3' cassette), while the 3' fragment islinked to a puromycin resistance gene (hprtΔ5' cassette). Cre-inducedrecombination between the loxP sites generates a fully-functional Hprtminigene which provides resistance to HAT selection in Hprt-deficientcells. The positive selectable markers are positioned so that followingrecombination, they are lost from the deleted chromosome. All of theclones that survive selection have the desired chromosomal structure. Asimilar positive selection system for detecting a chromosomaltranslocation has recently been reported by Smith et al., NatureGenetics 9:376-385 (1995). In addition, genes such as K14-agouti andtyrosinase genes can be preferably inserted into the vectors for use ascolor-coat markers, to aid in selecting the members of the populationfor which the chromosomal insert was successful. Albino mice lack thetyrosinase gene, so reinsertion of that gene is manifest by black micein a population of white mice. Similarly, the k14-agouti gene givesyellow color to the tips of the coat hairs against a black background(i.e., it makes black mice appear brown).

Initially, a small deletion (90 kb) was constructed which encompassesthe HoxB locus since the gene order and orientation was known.Subsequently, much larger chromosomal alterations were generated. Forthe latter alterations, knowledge of the transcriptional direction ofthe genes which serve as the rearrangement endpoints was not available.Consequently, it was necessary to generate ES cell lines with the fourpossible configurations of the hprt minigene fragments. Because thetranscriptional direction of the genes relative to the centromere wasalso unknown, it was not possible to predict which combination oforientations would give a deletion; however, clones with deletions arereadily distinguished in culture from the clones with other classes ofrecombinant chromosomes because in addition to becoming HAT resistant,both of the positive selection markers are lost. The generation of adeletion reveals the relative transcriptional direction of the twodeletion endpoints, and if the proximal-distal map positions are known(which was the case in these experiments), further deletions from thesame endpoint are greatly simplified.

The frequency of recombination between the loxP sites when they were onthe same chromosome varied from 6×10⁻⁷ to 5×10⁻⁵, but a directrelationship between the distance and the frequency was not apparent.The frequency of recombination was, however, significantly lower thanthose reported when the loxP sites are a few kb apart; see, Gu, et al.,Cell 73:1155-64 (1993), verifying that selection is required to isolatethese clones. These frequencies are derived by the transienttransfection of Cre in ES cells, but might be higher under conditions ofconstitutive expression of Cre, for example, in a specific lineage in atransgenic mouse. The frequency of recombination was reduced by one totwo orders of magnitude when the loxP sites were integrated in transcompared to the cis configuration. This is consistent with the knowledgethat individual chromosomes occupy discrete, non-overlapping domains inan interphase nucleus.

HAT-resistant clones derived from the trans configuration of the double-targeted clones oriented to give deletion products were not expected tobecome G418 or puro sensitive. But approximately half of theHAT-resistant clones segregated the puro cassette while all retained theneo cassette. This segregation pattern is consistent withinter-sister-chromatid recombination (see FIG. 4). Although the numberof clones with the trans configuration was relatively small, the equalratio of puro+neo to neo-only segregants suggests that G₂ recombinationis the predominant pathway used in this case. The rescue of hprtnegative daughter cells by metabolic cooperation also suggests that asubstantial fraction of the HAT-resistant clones derived from the cisdouble-targeted clones may have been generated by the sister-chromatidpathway.

The correlation of the induced chromosomal rearrangements with theorientation of the vectors has revealed physical mapping information inthis region of mouse chromosome 11. For instance, the genes described inthe following Examples, Gastrin, E2DH, Wnt 3 and the Hox B cluster, areall transcribed in the centromere-to-telomere direction. The E2DH-HoxBdeletion has shown that the HoxB cluster is oriented with the Hoxb-9gene nearest to the centromere.

EXAMPLE B Deletion and Duplication of 90 kb Containing the HoxB Cluster

The HoxB cluster provides an excellent substrate for the deletionstrategy since the cluster is about the same physical distance as a P1phage and the structure and orientation of the individual genes isknown. See, Rubock, et al., PNAS USA 87:4751-55 (1990). Moreover, adeletion allele of HoxB is very useful for detailed genetic analysis ofthis region.

To generate a HoxB deletion allele, the strategy outlined in FIG. 1 wasfollowed. The hprtΔ3' cassette was used to construct a targeting vectorfor Hoxb-1, the most 3' gene of the cluster, and targeted clones wereidentified (FIG. 2). An ES clone with the Hoxb-1 targeted allele (FIG.2B) was expanded and transfected with the Hoxb-9 targeting vectorcontaining the complementary hprtΔ5' cassette. The minigene fragmentswere oriented in the targeting vectors so that, after targeting, theywould be in the correct order and orientation with the positiveselection cassettes (neo and puro) located between the loxP sites (FIG.2C). Double-targeted clones were identified (FIG. 2C), and half of theseclones would be expected to have both targeted alleles on the samechromosome (cis) and half should have the targeted alleles on differenthomologues (trans).

To induce the recombination between the loxP sites, several independentdouble-targeted (Hoxb-1 and Hoxb-9) clones were expanded and transientlytransfected with a Cre expression cassette and placed under HATselection. Control transfections without Cre did not yield anyHAT-resistant clones. What follows is a more detailed description of themethod employed in this example.

As depicted in FIG. 1A, the PGKHprt minigene was modified by theinsertion of a loxP site from pBS64 (HindIII-EcoRI, Klenow blunt) intothe unique XbaI site (Klenow blunt) in the hprt intron. Insertion of theloxP site did not disrupt the cassette's HAT resistance function (notshown). The loxP-Hprt cassette was divided into two overlapping pieces:hprtΔ3', which contains the PGK promoter, the hprt exons 1 and 2, theloxP-intron, hprt exons 3-6, and the SV40 poly A signal. In FIG. 1A,hprtΔ5' and hprtΔ3' have a 2kb overlap including the loxP site;independently, hprtΔ5' and hprtΔ3' do not provide HAT resistance, butthey do when co-electroporated. HprtΔ5' and hprtΔ3' were ligated topositively selectable cassettes (hprtΔ3 ' to the pol II neo gene andhprtΔ5 ' to a PGK-puromycin resistance gene); in both cases, thepositive markers replaced the deleted part of the loxP-Hprt cassette,ensuring that, upon recombination, they are separated from thereconstituted cassette. FIG. 1C depicts the general strategy for makingdeletions consisting of 3 steps: Step 1: conventional replacement-typegene targeting used to replace the Hoxb-1 gene with the hprtΔ3 '-neocassette; Step 2: ES cells identified as correctly targeted are used asa substrate to insert the hprtΔ5 '-puro cassette into the endogenousHoxb-9 gene by conventional replacement-style targeting (the cisconfiguration is illustrated here); and Step 3: transient expression ofCre induces recombination between the loxP sites which reconstructs afunctional hprt minigene. In FIG. 1 C4, the intra-chromosomalrecombination pathway is illustrated, and in FIG. 1 C5, cells with therecombinant (deleted) chromosome to be positively selected in HAT mediaand a chromosomal ring are generated by the intra- chromosomal pathway.This is believed to be unstable and lost during the growth of thecolony.

The targeting vector for Hoxb-1 consists of a 3.5 kb BgIII-NcoI fragment(5' homologous arm); the Hoxb-1 coding sequence (1.7 kb Nco-BgIII) wasreplaced by the hprtΔ3 '-neo cassette and a 2 kb BgIII-PvuII fragment(3' homologous arm). The vector was linearized with SalI and a 10 μg waselectroporated into hprt-negative AB2.2 ES cells. G418 selection wasapplied 24 hours after the electroporation and resistant clones werearrayed in 96 well plates, and targeted clones were detected by Southernanalysis. A single targeted clone out of 384 clones analyzed wasidentified with the predicted structure of the targeted allele usingprobes 5' and 3' of the Hoxb-1 gene. This clone was expanded andtransfected with the Hoxb-9 targeting vector. The targeting vector forHoxb-9 consisted of a 6.2 kb HindIII fragment of homology which includedexon 1. The hprtΔ5'-puro cassette was cloned into the unique SalI sitein exon 1. The orientation of the cassette was such that, when targeted,the loxP sites in the hprtΔ cassettes would be in the same orientation.The vector was linearized and 10 μg of vector was electroporated intoclone #298 AB2.2 cells and plated on SNLP (puro resistant SNL76/7cells). Puromycin selection (5 μgnl) was applied 24 hours afterelectroporation. Resistant clones were arrayed in 96 well plates andscreened for targeted clones by Southern analysis. Double-targetedclones were detected at a frequency of 6%. Multiple independentdouble-targeted clones were expanded and independently transientlytransfected (by electroporation) with 20 μg of a supercoiled Creexpression cassette pOG231. HAT selection was applied 48 hours after theelectroporation. HAT-resistant clones were arrayed and analyzed bySouthern Blot analysis.

As evidenced by Table 1, two classes of double-targeted clones could bedistinguished by this assay.

                  TABLE 1                                                         ______________________________________                                                         Frequency (10.sup.-7)                                                         Deletion                                                     Interval     Distance      I    II  Inversion                                 ______________________________________                                        Hoxb9 - Hoxb1                                                                              90     kb     153  0.5 ND                                        Hoxb9 - E2DH 3-4    cM     6    1   43                                        Gastrin - E2DH                                                                             1      Mb     470  1.8 334                                       E2DH - Wnt3  3-4    cM     30   4   19                                        ______________________________________                                    

Table 1 reports the frequency of Cre-mediated recombination as afunction of distance between the loxP sites. Deletion frequencies areillustrated for both Class I and Class II clones while inversionfrequencies are only illustrated for Class I clones.

One class (Type I) yielded HAT-resistant recombinants at frequenciesaveraging 1×10⁻⁵ per treated cell, while a second class (Type II)yielded HAT-resistant clones at a much lower frequency.

Table 2 shows the frequency of Cre-mediated recombination as a functionof distance between the loxP sites.

                  TABLE 2                                                         ______________________________________                                        Example B                                                                     RECOMBINATION FREQUENCY ON                                                    THE MOUSE CHROMOSOME 11                                                                    Deletion                                                                              Inversion Duplication                                    Interval       Cis    Trans  Cis  Trans                                                                              Cis  Trans                             ______________________________________                                        Gastrin-E.sub.2 DH (1 Megabase)                                                              476    1      355  0    166  2                                 E.sub.2 DH-D11MIT199 (2 cM)                                                                  102    3      293  0    N/A  N/A                               HoxB-E.sub.2 DH (3-4 cM)                                                                     2      0      43   0    N/A  N/A                               E.sub.2 DH-Wnt3 (3-4 cM)                                                                     36     4      19   0    N/A  N/A                               E.sub.2 DH-D11MIT69 (22 cM)                                                                  0      0      3    0    N/A  N/A                               ______________________________________                                         (1 × 10.sup.7 ES cells were electroporated with 20 ug pOG 231 (Cre      expression plasmid) and were selected with HAT medium.)                  

Table 2 is similar to Table 1, except that the former shows anadditional deletion frequency for an additional interval (E₂DH-D11Mit69); it also shows duplication frequency; and finally it showsadditionally deletion, inversion, and duplication frequencies for bothcis- and trans-.

EXAMPLE C Cis and Trans Recombination

Two types of clones might correspond to the cis or trans configurationof the loxP sites. The HAT-resistant clones derived at a high frequencyfrom Type I clones might be products in intrachromosomal recombinationor sister chromatid exchange (loxP sites in cis). Type II clones mightrequire interchromosomal or inter-sister-chromatid recombination betweenhomologous chromosomes (loxP sites in trans), which may occur relativelyinfrequently. These different pathways were distinguished by analyzingthe markers in recombinant HAT-resistant clones.

Deletion of the HoxB cluster from a chromosome double targeted in ciswould be accompanied by the loss of the neo and puro cassettes. Theseare either segregated (sister-chromatid pathway) or a ring is formedwhich is presumed to be unstable (FIG. 1). The loss of both markerscould be documented in most of the HAT-resistant clones derived fromType I double-targeted clones, consistent with the hypothesized cisconfiguration of the Type I clones.

As anticipated, the consecutive targeting events and the Cre-inducedrecombination event results in the formation of novel restrictionfragments (FIG. 2). External probes identify the novel junctionfragments using an NheI digest since there is not an NheI site presentin the loxP-hprt cassette. An internal probe confirms the loss of 90 kbof sequence by dosage difference between the wildtype and the deletionclones (FIGS. 2E and F).

EXAMPLE D E2DH-Gastrin 1Mb Deletion

One of the goals behind the methods of the present invention is toconstruct chromosomal deletions so that regions of the genome can betested for tumor suppressor activity. Many candidate regions have beenidentified from loss of heterozygosity (LOH) studies. One well-definedregion in human breast cancer maps close to the Gastrin locus on humanchromosome 17q close to BRCA1. Miki, et al., Science 266:66-71 (1994).This putative sporadic tumor suppressor locus maps in a conservedlinkage group on mouse chromosome 11 between Gastrin and the E2DH locus.The generation of hemizygous mice with a deficiency that encompassesthis locus functionally tests if this region contains a sporadic breastcancer gene that is involved in mammary neoplasia.

The large size of the regions which contain putative sporadic tumorsuppressor loci complicates substantially the use of deletion strategy.In the absence of a YAC cloning contig which spans the relevant geneticinterval, the gene order and orientations were not known. This is animportant consideration since both the order and the orientation of theHprt minigene fragments will determine the type of chromosomalrearrangement that is required to reconstruct a functional Hprtcassette. The possible orientations are illustrated in FIG. 1D. Therecombinant chromosomes include deletions, duplications, inversions, anddi- and acentric chromosomes (FIG. 1E). These rearranged chromosomes canbe distinguished on Southern blots by the appearance of novel junctionfragments, but the most rapid identification of the clones withdeletions can be obtained from selection using neomycin and puromycinresistance cassettes which have been configured to lie between the loxPsites in the to-be-deleted interval (see FIG. 1).

To construct ES cell lines with large deletions between the Gastrin andthe E2DH locus (containing SBC I) in the absence of a priori knowledgeof the gene order and orientation, all four possible arrangements of thehprt minigene fragments were made and tested, only one of which willgenerate deletions. Two targeting vectors were constructed for eachdeletion endpoint representing the two possible orientations of the hprtminigenes. The hprtΔ3' cassette was targeted to the E2DH locus with thealternative minigene orientations (A or B). Targeted clones representingboth the A and B orientations were, in turn, transfected with thetargeting vectors representing the different orientations of the hprtΔ5'cassette (A or B) at the Gastrin locus. Multiple independent, targetedclones were isolated representing the four different minigeneconfigurations to ensure that clones with the cis and the transconfigurations were likely to be represented. Each of these clones wasexpanded, transfected with a Cre expression cassette and plated underHAT selection. What follows is a more detailed description of the methodemployed in this example.

Overlapping λ phage containing the mouse E2DH locus were isolated from amouse 129Sv/Ev genomic library using a human E2DH cDNA probe. Unlike theduplicated human E2DH locus, the mouse locus is present at a singlecopy. λ phage containing the mouse Gastrin locus were isolated from thesame library using a PCR fragment from the rat Gastrin cDNA. The mouseE2DH gene and Gastrin genes had not been mapped; to confirm that thesegenes mapped to mouse chromosome 11, a hamster/mouse hybrid cell line inwhich the mouse chromosome 11 is the only mouse genetic material washybridized to probes specific for the mouse Gastrin and E2DH loci.

Standard gene replacement targeting vectors were constructed from thesegenomic clones. E2DH vector: a total of 8.0 kb of homology was used. TheXhoI-XbaI 5.5 kb fragment containing the entire E2DH coding sequence wasreplaced with the hprtΔ3' minigene cassette in both orientations. In theGastrin vector, a total of 7.5 kb of homology was used. The 3.5 kbXhoI-NheI fragment containing the Gastrin coding region was replacedwith the hprtΔ5' cassette in both orientations.

The two vectors were separately transfected into AB2.2 ES cells. G418resistant clones were obtained for each vector. Clones were gridded onto96 well plates and screened for targeted clones. Targeted clones wereidentified at a ratio of 1/25 for the A orientation vector and 1/25 forthe B orientation vector. The two types of targeted ES cells wereassayed for totipotency by generating chimeras which tested for germline transmission. Totipotent E2DH-targeted ES cell lines wereidentified for both the A and the B orientation and these weretransfected with the vectors which target the Gastrin locus. Puromycinresistant clones were arrayed on 96 well plates and screened fortargeted clones. All four classes of double targeted clones wereobtained. For simplicity, this figure only shows the double targeted EScell having the hprtΔ5' and hprtΔ3' cassettes in the A orientation andin cis.

The double targeted ES cell clones were transfected with the Creexpression plasmid as previously described. HAT' colonies were recoveredand sibselected to test for puromycin and G418 resistance. Individualclones were expanded and analyzed for junction fragments using multipleprobes. Each blot was hybridized with two probes, one from the E2DHlocus and the other from the Gastrin locus. The frequency of obtainingHAT resistant colonies from the different clones is summarized in Table3.

                  TABLE 3                                                         ______________________________________                                                          Clones  m HAT.sup.r                                         Category Class    tested  (10.sup.7)                                                                            G418 Puro                                   ______________________________________                                        AA       I        4       470     S    S                                               II       2       1       R    R/S                                    AB       I        5       344     R    R                                               II       1       0       --   --                                     BA       I        3       377     R    R                                               II       2       0       --   --                                     BB       I        3       166     R    R                                               II       6       1.8     R/S  R                                      ______________________________________                                    

Table 3 reveals the frequency of the Cre-mediated recombination andretention of the markers in recombinant clones. All of the data isderived from the E2DH-Gastrin double targeted clones. The categories ofclones are illustrated in FIG. 1D, and the expected products aredescribed in FIG. 1E. Class I double-targeted clones give a highfrequency of HAT-resistant recombinants, while Class II clones give alow frequency of HAT-resistant clones. Retrospective analysis hasrevealed that the class I clones and Class II clones have the targetedgenes in cis and trans. S and R refer to resistance or sensitivity toG418 or puromycin as assayed by selection. Both resistant and sensitiveclones were recovered.

HAT-resistant clones were recovered from each of the four alternativesplit minigene configurations. The individual clones within a specificorientation group could be placed into one of two classes, based on thefrequency with which HAT-resistant clones could be recovered (Table 3).Selection analysis identified the AA class I HAT-resistant clones asthose that had lost the neomycin and puromycin resistance genes; theseclones are the most likely to have the desired deletion. Since the AAclass I clones gave the deletion product, this allows predictions to bemade on the likely products of the alternative configurations: BB givesduplications, and AB or BA should give inversions. These predictionshave been confirmed by detailed molecular analysis summarized in FIG. 3.In particular, the juxtaposition of the hprt minigene fragments whichwere previously positioned approximately 1 Mb apart in the genomeresults in unique junction fragments that are specific for the differenttypes of rearrangement.

The AA type II clones yield HAT-resistant recombinants at a lowfrequency. It was hypothesized that these clones represented the caseswhere the deletion selection cassettes had integrated in trans.Interchromosomal recombination would result in both the neo and puroresistance genes being located on one chromosome, while thereconstructed hprt minigene would be on the homologue. Thus it would beanticipated that all of the positive selection markers would be retainedin such a cell. Sibselection identified two classes of HAT-resistantclones which were represented at approximately equal frequencies. Onetype only retained the neo cassette, and a second type retained both theneo and the puromycin resistance cassettes (Table 1A). The segregationof the puro resistance gene from the neo cassette is explained readilyif Cre-induced recombination between sister chromatids (FIG. 4)occurred. This occurrence was confirmed by the molecular analysis ofthese clones. While the clones with the duplicated and deletedchromosomes can be generated by either interchromosomal or non-sisterchromatid exchange, the clones which only carry the neo cassette canonly have arisen by the non-sister chromatid recombination pathway.These clones have been confirmed to carry both the deletion chromosomeand the non-recombinant chromosome with only the E2DH targeted locus(FIG. 4).

The clones with the deletion on one chromosome and the duplication onthe other are genetically balanced. Therefor these clones wereconsidered to be the best candidates for germ line transmission. Fourindependent clones were injected into blastocysts, representing clonesdescended from both the A and B orientation of the E2DH targeted allele.Alleles from three of these clones were transmitted into the germ line,despite three cycles of subcloning and expansion. Segregation of thedeletion and duplication alleles from a chimeric male is illustrated bygene dosage analysis (FIG. 5). Mice which are hemizygous for thisdeletion (1 copy) are fully viable. Mice which are heterozygous for theduplication (3 copies) or homozygous (4 copies) are also fully viable(FIG. 5) and fertile.

EXAMPLE E Deletion of Two 34 Centimorgan Regions on Chromosome 11

Given the apparent insensitivity of the Cre-induced recombination to thedistance between the loxP-hprt substrates, two additional experimentswere performed to investigate if Cre could delete a larger fragment. Two3-4cM intervals were chosen, proximal or distal to the E2DH locus onmouse chromosome 11. This region is syntectic with a region on humanchromosome 17q where loss of heterozygosity studies have identifiedseveral distinct regions that are likely to contain tumor suppressorgenes which are mutated in 30-70% of sporadic breast cancer. Theseregions have been termed SBCI, SBCII and SBCII (FIG. 6A).

Since the E2DH-Gastrin deletion had revealed the orientation of the E2DHlocus, one of the A orientation E2DH-targeted clones was selected forthe proximal deletion and a B orientation E2DH-targeted clone wasselected for the distal deletion. Targeting vectors (two orientations)were constructed for the HoxB locus (Hoxb-9) and for Wnt3 (see Roelink,et al., PNAS USA 87:4519-23 (1990)). Double-targeted clones weregenerated, transfected with the Cre expression cassette andHAT-resistant clones were selected. One vector orientation yielded G418and puro sensitive clones which were hypothesized to have a 3-4 cMdeletion, while the other orientation yielded HAT-resistant clones whichall retain the neo and puro cassettes. This latter category of cloneswere confirmed to be inversions of the 3-4cM interval by molecularanalysis. The molecular analysis of the G418 andpuro sensitive clonesidentified two classes of clones that occur with approximately equalfrequency. The first type of clone was consistent with a simple deletionevent, illustrated for the Hoxb9-E2DH deletion (FIG. 6D). The otherclass of clone exhibited the expected deletion junction fragment, butalso retained a junction fragment that is diagnostic for the targetedchromosome, but should have been lost during the deletion event(illustrated for the E2DH-Wnt3 deletion in FIG. 6D). The retention ofthis junction fragment and the acquisition of the expecteddeletion-specific fragment in about half the clones can be explained bytwo different recombination pathways. The pure clones are believed to beproducts of an inter-chromosomal pathway (FIG. 1C), while those clonesthat retain the primary targeted allele may reflect a sister-chromatidexchange. While the duplicated chromosome should be segregated to adaughter cell and does not carry the reconstructed hprt minigene,extensive metabolic co-operation between the hprt⁺ and hprt⁻ ES cells ina colony facilitate cross rescue and substantial contribution of thehprt⁻ daughter cells to the HAT-resistant clones. What follows is a moredetailed description of the method employed in this example.

E2DH and Hoxb9 vectors have been described previously. Wnt3 genomicclones were isolated from a 129Sv/Ev genomic library using a cDNA probe.Conventional replacement targeting vectors containing 7.0 kb of homologywere constructed. The hprtΔ5' cassette replaces a 2.1 fragment (containsexons 3 and 4) of the Wnt3 gene. The Hoxb9 vectors and the Wnt3 vectorswere independently targeted into the E2DH targeted cell lines. An "A"orientation clone was used for the Hoxb9 targeting vectors. Transfectedcells were selected in puromycin and targeted clones were identified aspreviously described. Multiple independent double targeted clones weretransfected with the Cre recombinase. HAT-selection was used to isolaterecombinant clones which were sib-selected and tested by Southernanalysis for the predicted junction fragments.

EXAMPLE F Using a Virus Rather Than Targeting to Effect theRecombination

Rather than relying on traditional targeting techniques, either or bothof the desired deletion endpoints can be added to the genome by means ofa retrovirus. In this example, a viral vector is used to insert theendpoint at the 5' end only. FIG. 7 is a schematic representation of aprovirus structure suitable for this use, which is comprised of anhprtΔ5' minicassette, a loxP site and a puromycin resistance gene.Hence, the provirus structure is similar to the non-viral targetingvectors described in the previous examples.

In this example, only the 5' endpoint was inserted using the viralvector depicted in FIG. 7, though in other embodiments, both endpointscan be added using viral vectors. Chromosomal deletions were inducedusing the methods substantially as described in the previous examples.Hence, insertions are made at the two endpoints framing the desiredchromosomal deletion. The insertions are preferably made one at a time,and involve replacing a first native sequence (i.e., the first endpoint)on the chromosome of interest with a first selection cassette. Thisselection cassette consists of three elements: a first selectablemarker, a loxP site located in the hprt minigene intron, and a firstportion of a second selectable marker, preferably a non-functionalfragment of an Hprt minigene cassette. The first selectable marker ispreferably a neomycin resistance gene (hprtΔ5' cassette) or a puromycinresistance cassette (hprtΔ3' cassette). The cells expressing the marker(either the neomycin resistance gene or the puromycin resistance gene)are then selected. Next, the process is essentially repeated for secondendpoint on the chromosome of interest. Thus, the cells selected possessboth loxP sites framing the desired portion of the chromosome to bedeleted. The difference in this step is that the hprt minigenefragment--also non-functional--is the complementary portion to thatinserted into the first endpoint. Third, the selected cells arecontacted with Cre, which may be expressed in one of the three waysdescribed above, which induces recombination between the loxP sites.This recombination generates a fully functional Hprt minigene. Thisminigene provides resistance to HAT selection in hprt deficient cells.Additionally, the positive selectable markers are positioned so thatfollowing recombination, they are lost from the deleted chromosome.Therefore, the methods for inducing the deletion described in thisexample are nearly identical to those for practicing the method using anon-viral vector. Inherent differences in method and technique thatresult from using a viral versus non-viral vector are well-known to theskilled artisan, hence a detailed description of any embodiment of themethod of the present invention involving non-viral vectors could beeasily adapted by the skilled artisan using a viral vector. Therecombination efficiencies obtained from using the viral vector(described in FIG. 7) at the 5' endpoint are shown below in Table 4.

                  TABLE 4                                                         ______________________________________                                        # cell out of 10.sup.7 cre electroporated ES cells                                    HAT.sup.r                                                                          Puro.sup.r NO Drug  Rec Efficiency                               ______________________________________                                        plate #3  275    1.62 × 10.sup.5                                                                    3.9 × 10.sup.5                                                                 1.7 × 10.sup.-3                      plate #4  388    2.54 × 10.sup.5                                                                    3.9 × 10.sup.5                                                                 1.5 × 10.sup.-3                      Average   331    2.09 × 10.sup.5                                                                    3.9 × 10.sup.5                                                                 1.6 × 10.sup.-3                      ______________________________________                                    

EXAMPLE G Frequency of Cre-induced Deletion Between E₂ DH and D11 Mit199Using an Improved Targeting Vector

This example illustrates the enhanced Cre-induced deletion frequencyusing a different targeting vector compared with that used in theprevious examples. The details of the protocol are substantially asdescribed in the previous examples. Details of this "improved vector"compared with the vector used in the previous examples are shown inFIGS. 10 through 12 (the original vector is shown by comparison in FIG.12). FIG. 10 shows a map of an exemplary 5' endpoint targeting vectorautomatically excised out of a phage clone isolated from the 5' anchorlibrary. The 5' anchor library is shown in FIG. 9; this anchor librarycontains the expression cassettes 5' hprt, neomycin resistance gene, andtyrosinase gene. FIG. 11 shows a map of an exemplary 3' endpointtargeting vector automatically excised out of a phage clone isolatedfrom the 3' anchor library. The 3' anchor library is shown in FIG. 8;this anchor library contains the expression cassette 3' hprt, puromycinresistance gene, and k14-agouti gene. FIG. 12 shows a map of pG12WT(Wildtype 3' hprt cassette plasmid for making chromosomalrearrangements). At the bottom of FIG. 12, a comparison of the sequenceused to generate the data in Examples B-E versus the sequence used togenerate the data in this and following examples is shown. As evidencedby FIG. 12, the two sequences are identical to pG12 except that themutation in 3' hprt of the wildtype cassette plasmid has been fixed.

FIG. 13 shows the portion of the mouse chromosome 11 at which thedeletion strategy is directed; FIG. 13 also shows the generalcomposition of the selection cassettes positioned at the chromosomeendpoints, and the position of the Cre-induced deletion interval, E₂DH-D11Mit199.

Table 4 shows the frequency of Cre-induced deletion between E₂ DH andD11Mit199, which can be compared with Tables 1A, 1B, and 4. Thefrequency shown is the number of HAT-resistant colonies perCre-electroporated cell. The numbers are obtained by averaging data fromat least two experiments with at least two cell lines (except for thenew vector in the trans configuration). A comparison of the datapresented in Table 4 with those in Tables 1, 2, and 3 reveal that thecassette shown in FIG. 11 mediates recombination approximately 103 moreefficiently than the cassette used to generate the data in Tables 1, 2,and 4.

EXAMPLE H Frequency of Cre-induced Deletion Between E₂ DH and D11 Mit69Using an Improved Targeting Vector

Similar to Example H, this example also illustrates the enhancedCre-induced deletion frequency using a different targeting vectorcompared with that used in the previous examples. Example H illustratesthe Cre-induced deletion frequency between E₂ DH and D11Mit199-adistance of about 2 CM. By contrast, Example I illustrates theCre-induced deletion frequency between E₂ DH and D11Mit69-a distance ofabout 22 CM). The details of the protocol are substantially as describedin the previous examples. Details of this "improved vector" comparedwith the vector used in the previous examples are shown in FIGS. 10through 12 (the original vector is shown by comparison in FIG. 12). FIG.10 shows a map of an exemplary 5' endpoint targeting vectorautomatically excised out of a phage clone isolated from the 5' anchorlibrary. The 5' anchor library is shown in FIG. 9; this anchor librarycontains the expression cassettes 5' hprt, neomycin resistance gene, andtyrosinase gene. FIG. 11 shows a map of pG12WT (Wildtype 3' hprtcassette plasmid for making chromosomal rearrangements). The sequence isidentical to pG12 except that the mutation in 3' hprt has been fixed.

FIG. 14 shows the portion of the mouse chromosome 11 at which thedeletion strategy is directed; FIG. 14 also shows the generalcomposition of the selection cassettes positioned at the chromosomeendpoints, and the position of the Cre-induced deletion interval, E₂DH-D11Mit69. Table 6 shows the frequency of Cre-induced deletion betweenE₂ DH and D11Mit69, which can be compared with Tables 1A, 1B, and 4.

                  TABLE 6                                                         ______________________________________                                        Example H                                                                     Markers      Frequency                                                        ______________________________________                                        HAT.sup.r, Puro.sup.s, Neo.sup.2                                                           5.8 ± 3.3 × 10.sup.-6 (n = 5) / 2 × 10.sup.-5                  (n = 1)                                                          HAT.sup.r, Puro.sup.r, Neo.sup.r                                                           1.1 ± 0.4 × 10.sup.-5 (n = 10) / 3                                   × 10.sup.-5 (n = 1)                                        ______________________________________                                    

Two frequencies are reported in each row, reflecting two separatetrials. The "frequency" of Cre-induced loxP recombination is expressedas the number of HAT^(r) colonies per Cre-electroporated cell. Thenumber of independent doubly targeted cell lines is denoted by

                  TABLE 5                                                         ______________________________________                                        Example G                                                                     3' hprt cassette                                                                           Cis         Trans                                                ______________________________________                                        Old (mutant)   2 ± 0.5 × 10.sup.-5                                                            6.5 ± 3.2 × 10.sup.-7                       New (wildtype)                                                                             2.3 ± 1.3 × 10.sup.-2                                                            1.5 × 10.sup.-4                                ______________________________________                                    

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - <160> NUMBER OF SEQ ID NOS: 3                                               - <210> SEQ ID NO 1                                                           <211> LENGTH: 33                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Drosophila                                                    - <400> SEQUENCE: 1                                                           #         33       gatg tgatgaagga gat                                        - <210> SEQ ID NO 2                                                           <211> LENGTH: 35                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Human                                                         - <400> SEQUENCE: 2                                                           #       35         cgag atgtgagaag gagat                                      - <210> SEQ ID NO 3                                                           <211> LENGTH: 36                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Human                                                         - <400> SEQUENCE: 3                                                           #       36         cgag atgtgatgaa ggagat                                     __________________________________________________________________________

What is claimed is:
 1. A method for deleting a selected region ofgenetic material in cells comprising the steps of:inserting a firstselection cassette at a 5' end of said selected region usingconventional gene targeting methods, said first selection cassettecomprising a first selectable marker, a first loxP recombination site,and a first portion of a second selectable marker; selecting cellsexpressing said first selectable marker; inserting a second selectioncassette at a 3' end of said selected region using conventional genetargeting methods, said second selection cassette comprising a thirdselectable marker, a second loxP recombination site, and a remainingportion of said second selectable marker; expressing Cre recombinase toproduce recombination between said first and second loxP sites; andselecting cells expressing said second selectable marker, wherein theselected region of genetic material is deleted.
 2. The method of claim 1wherein said first selectable marker is a puromycin resistance gene. 3.The method of claim 1 wherein said second selectable marker is afunctional Hprt gene.
 4. The method of claim 1 wherein said thirdselectable marker is a neomycin resistance gene.
 5. The method of claim1 wherein said first selectable marker is a puromycin resistance gene,said second selectable marker is an Hprt gene, and said third selectablemarker is a neomycin resistance gene.
 6. The method of claim 1 or 2wherein said cells are embryonic stem cells.
 7. The method of claim 1 or2 wherein said cells are embryonic stem cells, and said cells aremaintained as cell lines.
 8. The method of claim 1 or 2 wherein said Creis transiently expressed Cre.
 9. The method of claim 1 or 2 wherein saidCre is inducibly expressed Cre.
 10. The method of claim 1 or 2 whereinsaid Cre is constitutively expressed Cre.
 11. A method of deleting aselected region of genetic material in cells comprising the stepsof:inserting a first selection cassette at a 5' end of said selectedregion using either a conventional gene targeting vector or a viralvector, said first selection cassette comprising a first selectablemarker, a first loxP recombination site, and a first portion of a secondselectable marker; selecting cells expressing said first selectablemarker; inserting a second selection cassette at a 3' end of saidselected region using a conventional gene targeting vector or a viralvector, said second selection cassette comprising a third selectablemarker, a second loxP recombination site, and a remaining portion ofsaid second selectable marker; expressing transiently Cre recombinase toproduce recombination between said first and second loxP sites; andselecting cells expressing said second selectable marker, wherein theselected region of genetic material is deleted.
 12. The method of claim11 wherein the viral vector is a retrovirus.
 13. The method of claim 11wherein the viral vector has a provirus structure comprising a cassettein turn comprising an hprtΔ5' cassette, a loxP site, and a puromycinresistance gene.
 14. The method of claim 11 wherein the viral vector hasa provirus structure comprising a cassette in turn comprising an hprtΔ5'cassette, a loxP site, and a neomycin resistance gene.
 15. The method ofclaim 11 wherein the targeting or viral vectors are a first vector forinserting a first native sequence of DNA at said 5' end, comprising:agenomic insert cloned into the vector of about 7.5 kb; a tyrosinaseminigene; a neomycin resistance gene; a 5' hprt fragment; and a loxPsite in a hprt fragment intron;and a second vector for inserting asecond native sequence of DNA at said 3' end, comprising: a genomicinsert cloned into the vector of about 8.5 kb; a K14-agouti gene; apuromycin resistance gene; a 3' hprt fragment; and a loxp site embeddedin a hprt fragment intron.
 16. The method of claim 15 wherein said 3'hprt fragment has the following SEQ. ID. NO. 1 at the start of exon 3:GAC,TGA,ACG,TCT,TCG,AGA,TGT,GAT, GAA,GGA,GAT.
 17. A replacement vectorsystem comprising:a first vector for inserting a first native sequenceof DNA at said 5' end, comprising:a genomic insert cloned into thevector of about 7.5 kb; a tyrosinase minigene; a neomycin resistancegene; a 5' hprt fragment; and a loxP site in a hprt fragment intron; anda second vector for inserting a second native sequence of DNA at said 3'end, comprising:a genomic insert cloned into the vector of about 8.5 kb;a K14-agouti gene; a puromycin resistance gene; a 3' hprt fragment; anda loxP site embedded in a hprt fragment intron.
 18. The vector system ofclaim 17 wherein said 3' hprt fragment has the following sequence [SEQ.ID. NO. 1] at the start of exon 3: GAC,TGA,ACG,TCT,TCG,AGA,TGT,GAT,GAA,GGA,GAT.
 19. A method for creating defined chromosomaldeficiencies, deletions, and duplications comprising the stepsof:identifying a desired region of a chromosome of interest to bedeleted; inserting two native sequences at each endpoint of said regionof said chromosome of interest using a first and a second targetingvector, each comprised of one or more selectable markers and a loxP siteand an hprt fragment; transiently expressing Cre recombinase to producerecombination between each of two said loxP sites; whereby uponchromosomal rearrangement induced by said Cre recombinase, a functionalHprt expression cassette is reconstructed, wherein defined chromosomaldeficiencies deletions and duplications are created.